1. Field
The present disclosure relates to a method of amplifying RNA from a sample and a method of amplifying a pool of RNAs in a sample.
2. Description of the Related Art
Amplification of a small amount of RNA obtained from a specific biological sample can be the most fundamental and important step in a genetic analysis of the sample. Transcriptome analytic methods, quantitative analysis of RNA (e.g., gene-expression analysis) and qualitative analysis of RNA (e.g., RNA-sequence analysis) are the main methods used to analyze RNA from biological tissues. In each of these methods, amplifying the RNA to a sufficient amount without modification allows for further accurate and meaningful analysis.
A conventional method of amplifying an RNA pool for transcriptome analysis includes reverse transcribing linear RNA to produce single-stranded DNA using oligo dT primers or primers with random sequences, displacing the single-stranded DNA with a double-stranded DNA using random primers, ligating different adapters to a 5′-end and a 3′-end of the double-stranded DNA, and preparing a complementary DNA (cDNA) library by amplifying the DNA.
According to the conventional method, cDNA conversion efficiency may differ depending on the RNA used in the reverse transcription process because, for example, the RNA may have various primary or secondary structures. Also, reverse transcribed DNA corresponding to some expressed genes may be lost due to improper adapter ligation.
Moreover, the conventional method may require reversing the sequence of amplified RNA (or cDNA) to obtain the original RNA sequence direction after sequence analysis because the ligated adapters cannot recognize the directionality(i.e., the adapters do not distinguish between the reverse transcribed cDNA ends corresponding to the 5′-ends and 3′-ends) of the original RNA.
In addition, amplification bias may occur during an amplification process of RNA according to the conventional method. In particular, a polymerase chain reaction (PCR) using a specific sequence may result in biased amplification of some reverse transcribed target DNAs more than others, depending on the sizes and characteristics of the target sequences. A multiple displacement amplification (MDA) using primers of random sequences may also result in different bonding efficiencies of primers depending on the target(s) due to the diversity of structures and sequences of the single-stranded DNAs. Therefore, amplification bias resulting in different degrees of amplification of different genes may be observed in conventional MDA methods.
Methods are desired to reduce loss of genes and/or amplification bias during preparation of cDNA libraries. Methods are desired that allow directionality of the original RNA to be retained in the amplified RNA.